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Letter

Design and assembly of DNA sequence libraries for chromosomal insertion in bacteria based on a set of modified MoClo vectors Daniel Schindler, Sarah Milbredt, Theodor Sperlea, and Torsten Waldminghaus ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00089 • Publication Date (Web): 15 Jun 2016 Downloaded from http://pubs.acs.org on June 21, 2016

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ACS Synthetic Biology

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Manuscript for ACS Synthetic Biology

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Design and assembly of DNA sequence libraries for chromosomal insertion in

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bacteria based on a set of modified MoClo vectors

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Daniel Schindler‡, Sarah Milbredt‡, Theodor Sperlea‡, Torsten Waldminghaus*

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‡equal contribution

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Chromosome Biology Group, LOEWE Center for Synthetic Microbiology, SYNMIKRO, Philipps-

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Universität Marburg, Hans-Meerwein-Str. 6, D-35043 Marburg, Germany

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Abstract

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Efficient assembly of large DNA constructs is a key technology in synthetic biology. One of the most

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popular assembly systems is the MoClo standard in which restriction and ligation of multiple

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fragments occurs in a one-pot reaction. The system is based on a smart vector design and type IIs

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restriction enzymes which cut outside their recognition site. While the initial MoClo vectors had been

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developed for the assembly of multiple transcription units of plants, some derivatives of the vectors

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have been developed over the last years. Here we present a new set of MoClo vectors kit for the

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assembly of fragment libraries and insertion of constructs into bacterial chromosomes. The vectors

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are accompanied by a computer program that generates a degenerate synthetic DNA sequence that

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excludes ‘forbidden’ DNA motifs. We demonstrate the usability of the new approach by construction

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of a stable fluorescence repressor operator system (FROS).

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Keywords

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genome engineering; chromosome; software; Escherichia coli; sequence design; synthetic biology

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Introduction

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Biotechnology as well as basic research in biology often includes changing the organism of interest. In

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some cases, one might want to teach microorganisms to produce some valuable chemical, in other

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cases one wants to see the effect of additional factors or how cells compete without a certain

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component. Thus, the ability to introduce changes in an efficient way is a key for future life science

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developments. Alterations of organisms will, in most cases, be made on the DNA level from which

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the phenotypic characteristics are derived. The development of genetic modification started in the

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1970 with the first recombinant DNA being used to transform cells and has since been extended

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enormously. Especially the research field of synthetic biology came along with a multitude of new

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techniques for DNA manipulation and assembly (1-5). These new DNA assembly approaches were

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developed to overcome certain limitations of traditional cloning strategies. One major issue is that

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cloning based on DNA ligase and regular restriction endonucleases often leaves the respective cut

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sites as scar in the assembled product. However, there are at least four DNA assembly approaches

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for scar-free assembly of DNA fragments (1-3). First, the Gibson assembly is based on homologous

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ends of DNA fragments, which are fused in an in vitro reaction including an exonuclease, a DNA

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polymerase and a DNA ligase (5). This is similar to the second approach where the homologous ends

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are fused in vivo by the highly efficient recombination system of the yeast Saccharomyces cerevisiae

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(6). In a third approach, the ligase cycling reaction (LCR), the homology is not mediated by the DNA

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fragment ends but by a bridging oligonucleotide (7). A fourth approach makes use of type IIs

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restriction enzymes (8). These enzymes are distinct from other restriction enzymes in that they cut

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outside their recognition site. They are directional and the positioning of the recognition site allows

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determination where the DNA is cut. Notably, the actual cut site can be freely chosen allowing the

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design of scar-less assemblies.

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An important benefit of the four described methods as compared to traditional cloning is their

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suitability for fast, single reaction multi-fragment assembly. The first three approaches are

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dependent on homologous regions of about 20-40 bps which will determine the position of

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fragments in a multi-part assembly. With type IIs restriction sites the required homology is limited to

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only 4 bps. This fact was used to develop hierarchical assembly systems based on vectors with

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defined 4 bp sequences to fit one another (8-10). Such a system allows the efficient assembly of

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many fragments into a destination vector independent of the actual sub-fragment sequence or size.

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Probably the most popular type IIs-based assembly framework is the MoClo system developed by

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Sylvestre Marillonnet and colleagues (9, 11). It consists of sets of seven vectors with the 4 bp

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overhang ends of each vector matching the overhangs of the preceding and following vector,

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respectively. Assembling fragments from one vector set (one level) into the next is possible because

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the resistance markers as well as the type IIS restriction enzymes and sites are alternating. A set of

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endlinkers is used to generate matching ends for assembly of different numbers of fragments into

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one acceptor vector (9, 11).

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One important benefit of the MoClo approach is that it is based on mixing complete plasmids

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eliminating the need for PCR or fragment isolation. Recently, the MoClo system was adapted to or

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optimized for special purposes as transcription unit assembly in plants, mammals, fungi or bacteria

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(12-15). Here we present modifications of the MoClo system for efficient cloning of sequence

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libraries and for the construction of fragments to be inserted into the E. coli chromosome. We

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introduce a computer tool for sequence design and show the feasibility of our approach by designing

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and assembling a FROS array (fluorescence repressor operator system).


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FROS is a widely used tool for spatial and temporal visualization of genetic loci in vivo and has been

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applied to various different organisms (16-18). Fluorescently labelled DNA binding proteins are used

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to highlight specific binding sites, which are integrated at a gene locus of interest by homologous

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recombination. It was initially applied with tandem repeats of the lac operator and a gfp fused Lac

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repressor in yeast and CHO-cells (16). Also a tet operator-based FROS system was generated and

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used in yeast (19). As the transfer of FROS to bacteria was not very successful due to instability

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caused by large homologous regions; arrays were optimized by insertion of random spacers in

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between the operator repeats to decrease homology (20, 21). As further improvement the number

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of binding sites can be reduced from 250 to 64 to limit interference with the replication machinery

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(22). FROS was subsequently applied successfully in various bacteria to gain new insights into the

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localization, replication and segregation of chromosomes (17, 23, 24). Nevertheless, the design and

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generation of DNA sequences with many repetitive elements remains challenging. In this paper we

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present a new set of MoClo vectors that allowed generation of a FROS array with 64 binding sites of

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two different operators in just 4 cloning steps based on a single pair of degenerate oligonucleotides

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and its subsequent integration into the chromosome of E. coli.

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Results and discussion

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A computer tool to generate sequences with restricted diversity

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Efficient assembly of DNA fragments is critical for modern molecular biology approaches. It was

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predicted that software tools will have an increasing importance for DNA assembly approaches (2).

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Often, sequences are needed that have specific DNA motifs at defined sites but not at others. Other

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DNA motifs, as for example restriction sites, need to be excluded throughout the whole construct. It

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might be straight forward to design a single exact sequence with these characteristics based on

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extension of two DNA oligonucleotides with an overlap region at one end (Fig. 1A). However,

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efficient cloning strategies should allow working with libraries generated from mixtures of DNA

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oligonucleotides to lower the overall costs. Here we present the computer program MARSeG (Motif

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Avoiding Randomized Sequence Generator) that generates degenerated sequences with a high

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degree of diversity while excluding a list of DNA motifs provided by the user (Fig. 1A). An example for

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its application could be the design of 20 spacer sequences with a length of 200 bps each, that are

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used to separate transcription units within a large scale gene circuit assembly. Notably, these spacer

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sequences should not harbor recognition sites for a list of restriction enzymes. Instead of designing

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and buying 20 individual sequences one could just order a fully randomized sequence with 200 Ns

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and receive an oligonucleotide mix to be cloned into a vector backbone. However, a certain amount

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of these sequences will have at least one of the ‘forbidden’ DNA motifs. We tested this assumption


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by comparing random sequences with sequences generated with MARSeG (Fig. 1B). Almost 60 % of

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completely random sequences with a length of 200 bps contain at least one motif from a list of 19

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restriction enzyme recognition sites (motif list III in table S1, fig. 1B). The computer tool MARSeG

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reduces the diversity of sequences in such a way that ‘forbidden’ motifs are excluded while

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maintaining high sequence diversity. This leads to sequence collections without any appearance of

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the ‘forbidden’ motifs (Fig. 1B). The degree of MARSeG library diversity will depend on the number

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and type of DNA motifs to be excluded as shown by analysis of the overall sequence homology for

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100 example sequences for three different lists with two, ten or nineteen ‘forbidden’ DNA motifs,

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respectively (Table S1, fig. 1C). An alternative approach would be to generate many sequences of the

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desired length and exclude all sequences that do contain one or more of the ‘forbidden’ motifs or

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other undesired characteristics (25). However, this approach will only generate individual sequences

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and no sequence libraries as MARSeG does. MARSeG is open source and available, including a

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detailed user manual, through the web site (http://www.synmikro.com/marseg).

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A new set of MoClo vectors optimized for library cloning and insertion into the E. coli chromosome

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The MoClo vectors are widely used and some specialized derivatives or part libraries have been

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developed (12, 13, 26). We changed the existing vectors to facilitate library cloning, multi-fragment

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assembly and insertion of constructs into bacterial chromosomes via homologous recombination

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techniques. An overview of the modifications is depicted in figure 2A and a list of new vector sets is

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given in table S2. The starting point for our modifications was a set of MoClo vectors kindly provided

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by Sylvestre Marillonnet. The respective Level 1 vectors have been described previously and the level

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M and P vectors differ from previous vectors by the fact that they do not contain T-DNA borders for

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agrobacterium delivery (9). Working with libraries instead of individual sequences poses special

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requirements on the DNA assembly system. Most importantly, the percentage of positive clones

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should be near 100% because clones are not selected individually. To suppress vectors still containing

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the lacZ cassette instead of the desired fragment, we added ccdB gene in such a way that it is lost

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with the lacZ gene upon successful cloning (ccdB+ vectors). The ccdB gene product is a small

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cytotoxin that kills E. coli cells that are not engineered to express the antitoxin CcdA or possess a

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mutated gyrase (27). As expected, cloning with the ccdB+ vector led to elimination of the blue

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colonies still harboring the lacZ-ccdB MoClo cassette (Fig. 2B).

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A second change to previous MoClo vectors is a size reduction of the sequence remaining between

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level 1 fragments in higher level assemblies. Respective sequences where placed in the original level

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1 vectors between the BpiI and BsaI sites and contain restriction sites to facilitate the analysis of

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assembled transcription sites. For this purpose they were certainly helpful but could be deleterious in

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other cases for example as potential recombination sites if occurring to frequently. To keep this short


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sequences remaining between the assembled fragments as small as possible we deleted the 12 bp

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between the BsaI and the BpiI cut sites for the whole level 1 vector set.

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Very often it is desired to introduce constructed gene circuits or pathways into the host chromosome

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as the genomic stability is higher compared to plasmid based expression (28). In addition, the cell to

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cell variability of plasmid copy numbers makes it difficult to derive quantitative data for exact

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measurements of expression phenomena (29). Chromosomal insertions into the E. coli chromosome

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are straight forward with the phage lambda based recombination system (30). However, a frequent

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problem are false-positives originating from transferred plasmids even if those just served as PCR

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template or were supposed to be cut by restriction enzymes. To eliminate this problem we

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exchanged the original pMB1 replication origin with oriR6K. This conditional replication origin does

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only replicate in E. coli strains expressing the lambda pir gene and thus, replicons based on oriR6K are

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not able to replicate in wildtype E. coli. As a proof of principle we cloned building blocks for a

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chromosomal insertion into level 1 vectors, including homologous regions targeting the lac locus, a

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chloramphenicol resistance marker flanked by FRT sites to remove the cassette after successful

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integration via ‘flipping’ and a fluorescence gene with a constitutive promoter. After a one-step

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assembly of all four parts into one of our new vectors the assembled construct could readily be

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inserted into the E. coli chromosome by recombineering to generate red fluorescent cells (data not

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shown). All vectors as well as the parts described here and below (72 plasmids in total) are available

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through a request form on our homepage (http://www.synmikro.com/plasmidrequest). An overview

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of the new MoClo vectors and their position within the MoClo hierarchy is shown in supplementary

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figure S1.

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Application of new vectors to construct a repressor-operator array

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To test the usability of the MARSeG program and the new MoClo vectors we applied these tools to a

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more challenging assembly, namely the construction of a FROS system. Such systems consist of an

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array of operator sites which are bound by a fluorescence marker fused to the respective repressor

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protein to visualize a specific genomic region by microscopy. These arrays are difficult to assemble

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because the operator sequences are homologous to one another. Such repetitive sequences have

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been shown to be especially difficult to assemble with methods relying on larger homology parts as

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Gibson assembly (8, 31). The array we designed contains tet as well as lac operators to allow more

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flexibility in the choice of binding proteins. Construction of a FROS array of 128 operators (64 TetO

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plus 64 LacO) was based on building blocks with 8 alternating operator sequences separated by

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variable linker sequences to reduce homology between building blocks (Fig. 3A). The basic building

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blocks were generated by elongation of two overlapping DNA oligonucleotides designed with

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MARSeG (Fig. 3A, see Method section for details). Fragment libraries were applied to a MoClo


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reaction with seven level 1 vectors (Fig. 3A). After transformation of the cloning reaction the

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generated plasmid libraries were directly purified from the liquid E. coli culture and used for a four

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part assembly into level M vectors (Fig. 3A). Each of these parts should contain 32 operators with

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each of the operators being separated by a different spacer sequence of different length which was

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designed by MARSeG to be diverse on one hand but to not contain recognition sites of the type IIs

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endonucleases used (Fig. 3A). To test this diversity, we sequenced the operator array regions after

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the second assembly step (pMA281-284, see supl. table S4) and aligned all spacer sequences with

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one another. The respective homology matrix is shown in figure 3B. Notably, none of the 155 spacer

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sequences appeared more than once in the array. Homologies ranged between 0 and 90 %, clearly

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showing that the design and cloning approach presented here is able to produce a suitable amount

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of sequence diversity.

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To further test the functionality of the constructed array it was integrated into the E. coli

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chromosome via the new vector system as described above. Cells carrying this integration were

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transformed with a plasmid allowing inducible expression of the fluorescence protein mVenus fused

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to the TetR repressor. Fluorescence microscopy showed clear formation of foci in cells with the

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constructed FROS array insertion as expected (Fig. 4A). In contrast, only diffuse fluorescence was

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seen in cells lacking the FROS array (Fig. 4A). These results demonstrate the functionality of the

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constructed FROS array. A common problem with FROS arrays in which the spacer sequences

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between the operators have the same sequence and are not diverse as in our case is their genetic

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instability caused by homologous recombination events. This can lead to undesired size reduction of

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the respective FROS array. To test if the FROS array presented here is resistant to such recombination

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events we cultured the E. coli strain carrying the array for an extended period of 120 h (see Material

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section for details). After 24h periods we measured the array size by Southern Blotting (Fig. 4B). No

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fragments smaller than the expected 11247 bps were detected over the entire test period supporting

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genetic stability of the constructed FROS array (Fig. 4B). To further test if the genetic stability of the

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FROS array with MARSeG-based design outperforms that of an array with the same operator setup

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but similar instead of diverse spacer sequences we constructed such a “bad-design-array” with 128

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operator sites as above. We cultivated the respective plasmid pMA704 in E. coli MG1655

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continuously for several days in parallel to cells carrying a similar plasmid with the MARSeG-designed

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spacers (pMA290). The plasmid DNA was isolated after 24 hour intervals and cut with BpiI to release

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the 4817 bp FROS array. A respective band can be seen at all analyzed time points for the FROS array

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with MARSeG design (Fig. 4C, left). In contrast the FROS array band becomes weaker starting at 48h

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of cultivation in case of the similar spacer sequences (Fig. 4C, right). In addition, smaller bands occur

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on the agarose gel at later time points, clearly indicating plasmid size reduction through homologous

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recombination. We conclude that the FROS array designed using MARSeG has a higher genetic


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stability as an array with all spacer sequences being similar. It is important to note that stable FROS

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arrays with variable linkers have been constructed before (17). However, the assembly approach

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presented here presents a threefold improvement to the earlier work. First, the MARSeG design

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excludes unwanted restriction sites to omit unwanted cloning of erroneously cut sub-fragments

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instead of full fragments. Second, the previous assembly approach required laborious purification of

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DNA fragments for cloning instead of the plasmid-based MoClo approach used here. Third, the

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previous approach included a doubling of operator sites in each assembly step while the MoClo

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hierarchy used here generates a four-fold increase of sites in each step. This reduces the number of

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cloning steps which will be more important the bigger the assembly of interest is.

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Chromosomal insertions into the lac operon are a common approach but are limited to E. coli strains

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that actually carry this gene region. To allow more flexibility and potentially target multiple

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chromosomal sites we have designed and constructed flanking regions for five additional

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chromosomal loci in the new MoClo vectors (Suppl. table S4, suppl. fig. S2). We have used respective

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vectors to assemble a cassette targeting tnaA and could successfully use it for insertion of the FROS

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array (32)(Suppl. table S3 and S4). As for the integration into lacZ we observed fluorescence foci

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showing functional chromosomal integration (data not shown).

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Conclusions

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The ability to efficiently assemble DNA constructs and integrate them into a host genome is still a

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main bottleneck in basic and applied molecular biology research. New methods have been developed

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over the last years allowing multi-fragment assembly based on different principles. The next step

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must be the adaptation and optimization of these new approaches to specific systems. Here we

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present tools for the design and efficient multi-fragment assembly of genetic constructs for

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chromosomal insertion. Our new MoClo vectors are fully compatible with previously published

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MoClo kits of the Marillonnet group. Although we focus on manipulation of the E. coli chromosome

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our approach should be applicable in many bacteria that allow genetic modification via homologous

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recombination. We expect the approach presented here to be especially valuable for the design and

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construction of synthetic chromosomes which is now technically possible (1, 4, 5, 33).

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Material and Methods

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A detailed description of the materials and methods is provided in the supplementary material.

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Author Contribution

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DS, SM and TS contributed equally to this work.

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Acknowledgments ACS Paragon Plus Environment

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We gratefully thank Sylvestre Marillonnet (Halle, Germany) for providing MoClo vectors and helpful

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discussions. Johan Elf (Uppsala, Sweden), Federico Katzen and Xiquan Liang (Thermo Fisher Scientific,

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Carlsbad, USA), Alexander Böhm (Marburg, Germany, †), William Margolin (Houston, USA) and

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Michael L. Kahn (Washington, USA) are acknowledged for providing strains and/or plasmids. We

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thank Julian Sohl, Joel Eichmann and Patrick Sobetzko from the Waldminghaus lab for helping with

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experiments and data analysis as well as Nadine Schallopp for excellent technical assistance and the

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whole working group for fruitful discussions. We are grateful to Manuel Seip for help with setting up

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the web pages. This work was supported within the LOEWE program of the State of Hesse.

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Supporting Information - Fig. S1: New vectors and their hierarchy within the MoClo system. - Fig. S2: Insertion sites on the E. coli chromosome to be targeted with the new MoClo vectors. - Supplementary Methods. - Tables S1: DNA motifs to be excluded in sequences designed by MARSeG. - Table S2: Overview of optimized MoClo plasmids and their respective changes. - Table S3: Strains used in this study. - Table S4: Plasmids used in this study. - Table S5: Oligonucleotides used in this study.

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Figure 1: Generation of diverse DNA sequences that exclude of a list of motifs by MARSeG. (A)

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Double-stranded DNA fragment libraries are generated by annealing and extension of two single-

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stranded oligonucleotides (black lines) with partial overlap (gray boxes). Three possible designs are

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shown below. A fully defined sequence (first line) could exclude a list of motifs but does not confer

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diversity; a fully randomized sequence (second line) confers diversity but might lead to sequences

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including unwanted motifs. Sequences generated with MARSeG (third line) confer diversity while

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excluding unwanted motifs. (B) Sequences of 200 bps were generated completely random (red) or

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with MARSeG (blue) and the number of motifs from a list of 19 ‘forbidden’ restriction enzyme

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recognition sites (list III in table S1) was counted for 500 derived sequences. (C) Trade-off between

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the amount of excluded motifs and diversity in MARSeG generated sequences. After generating

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degenerate sequences using MARSeG with three motif lists as indicated (Tab. S1), 100 sequences

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were defined from each respective template. Pairwise sequence homology values were calculated

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using a Smith-Waterman algorithm. The degree of homology is color coded as indicated.

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Figure 2: Optimization of MoClo vectors for library cloning and chromosomal insertions. (A)

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Schematic drawing of changes to existing MoClo vectors: arrows = insertion, double arrows =

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exchange, red cross = deletion. (B) Cloning into standard MoClo vectors produces some background

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consisting of original vectors, indicated by blue colonies (top panel). New vectors including ccdB lead

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to white colonies only (bottom panel). Percentage of colonies is given in the respective color.

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Figure 3: Assembly of the LacO/TetO operator array. (A) DNA oligonucleotides were annealed (gray

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boxes), elongated and enriched via PCR. Linker lengths (in bps) are shown as white numbers. The

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resulting library was cloned into seven level 1 vectors. Sets of four level 1 vector libraries were

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assembled into level M acceptor vectors and four resulting individual vectors were combined into

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level P to gain the final array. For integration into E. coli lacZ, flanking regions and a chloramphenicol

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cassette (flanked by FRT sites) were assembled together with the final array into level M. (B) Spacer

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sequence homology matrix. The sequenced FROS array assembly parts (pMA281-284, see supl. table

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S4) were disassembled and the pairwise homologies of spacer sequences were calculated using a

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Smith-Waterman algorithm. The respective homology is color coded as indicated.

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Figure 4: In vivo functionality of the constructed FROS array. (A) Fluorescence microscopy of E. coli

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cells harboring a plasmid encoding a TetR-mVenus fusion and either no FROS array (top panel; strain

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SM100) or a chromosomal integration of the new FROS array (bottom panel; strain SM112). The scale

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bar is 2 µm. (B) Southern Blot analysis to test stability of the LacO/TetO array during extended

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cultivation. Chromosomal DNA was isolated from strain SM93 after different time points of

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cultivation as indicated and cut with NdeI. DNA was plotted on a membrane after separation on an

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agarose gel and the array detected with a probe directed against lacI. Black asterisk highlights the

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size of the array (11247 bps). As control we used DNA from wildtype E. coli MG1655 without FROS

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integration resulting in a fragment of 7520 bps. (C) Genetic stability of a FROS array with MARSeG-

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designed variable spacer sequences (left) compared to an equivalent array with each spacer

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sequence being similar to one another. E. coli strains DS366 und DS367 carrying plasmids pMA290

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and pMA704 respectively were cultivated for the indicated time periods. Plasmid DNA was isolated

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and cut with BpiI to release the array (4817 bp) and the vector backbone (3968 bp) as indicated. ACS Paragon Plus Environment

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References

332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382

1. 2. 3.

4.

5.

6. 7.

8. 9. 10.

11.

12. 13.

14.

15.

Schindler, D., and Waldminghaus, T. (2015) Synthetic chromosomes, FEMS microbiology reviews 39, 871-891. Casini, A., Storch, M., Baldwin, G. S., and Ellis, T. (2015) Bricks and blueprints: methods and standards for DNA assembly, Nat Rev Mol Cell Biol 16, 568-576. Karas, B. J., Suzuki, Y., and Weyman, P. D. (2015) Strategies for cloning and manipulating natural and synthetic chromosomes, Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology 23, 57-68. Annaluru, N., Muller, H., Mitchell, L. A., Ramalingam, S., Stracquadanio, G., Richardson, S. M., Dymond, J. S., Kuang, Z., Scheifele, L. Z., Cooper, E. M., Cai, Y., Zeller, K., Agmon, N., Han, J. S., Hadjithomas, M., Tullman, J., Caravelli, K., Cirelli, K., Guo, Z., London, V., Yeluru, A., Murugan, S., Kandavelou, K., Agier, N., Fischer, G., Yang, K., Martin, J. A., Bilgel, M., Bohutski, P., Boulier, K. M., Capaldo, B. J., Chang, J., Charoen, K., Choi, W. J., Deng, P., DiCarlo, J. E., Doong, J., Dunn, J., Feinberg, J. I., Fernandez, C., Floria, C. E., Gladowski, D., Hadidi, P., Ishizuka, I., Jabbari, J., Lau, C. Y., Lee, P. A., Li, S., Lin, D., Linder, M. E., Ling, J., Liu, J., London, M., Ma, H., Mao, J., McDade, J. E., McMillan, A., Moore, A. M., Oh, W. C., Ouyang, Y., Patel, R., Paul, M., Paulsen, L. C., Qiu, J., Rhee, A., Rubashkin, M. G., Soh, I. Y., Sotuyo, N. E., Srinivas, V., Suarez, A., Wong, A., Wong, R., Xie, W. R., Xu, Y., Yu, A. T., Koszul, R., Bader, J. S., Boeke, J. D., and Chandrasegaran, S. (2014) Total synthesis of a functional designer eukaryotic chromosome, Science 344, 55-58. Gibson, D. G., Glass, J. I., Lartigue, C., Noskov, V. N., Chuang, R. Y., Algire, M. A., Benders, G. A., Montague, M. G., Ma, L., Moodie, M. M., Merryman, C., Vashee, S., Krishnakumar, R., Assad-Garcia, N., Andrews-Pfannkoch, C., Denisova, E. A., Young, L., Qi, Z. Q., Segall-Shapiro, T. H., Calvey, C. H., Parmar, P. P., Hutchison, C. A., 3rd, Smith, H. O., and Venter, J. C. (2010) Creation of a bacterial cell controlled by a chemically synthesized genome, Science 329, 5256. Ma, H., Kunes, S., Schatz, P. J., and Botstein, D. (1987) Plasmid construction by homologous recombination in yeast, Gene 58, 201-216. de Kok, S., Stanton, L. H., Slaby, T., Durot, M., Holmes, V. F., Patel, K. G., Platt, D., Shapland, E. B., Serber, Z., Dean, J., Newman, J. D., and Chandran, S. S. (2014) Rapid and reliable DNA assembly via ligase cycling reaction, ACS synthetic biology 3, 97-106. Engler, C., Kandzia, R., and Marillonnet, S. (2008) A one pot, one step, precision cloning method with high throughput capability, PLoS One 3, e3647. Weber, E., Engler, C., Gruetzner, R., Werner, S., and Marillonnet, S. (2011) A modular cloning system for standardized assembly of multigene constructs, PLoS One 6, e16765. Storch, M., Casini, A., Mackrow, B., Fleming, T., Trewhitt, H., Ellis, T., and Baldwin, G. S. (2015) BASIC: A New Biopart Assembly Standard for Idempotent Cloning Provides Accurate, Single-Tier DNA Assembly for Synthetic Biology, ACS synthetic biology 4, 781-787. Werner, S., Engler, C., Weber, E., Gruetzner, R., and Marillonnet, S. (2012) Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system, Bioengineered bugs 3, 38-43. Lee, M. E., DeLoache, W. C., Cervantes, B., and Dueber, J. E. (2015) A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly, ACS synthetic biology 4, 975-986. Engler, C., Youles, M., Gruetzner, R., Ehnert, T. M., Werner, S., Jones, J. D., Patron, N. J., and Marillonnet, S. (2014) A golden gate modular cloning toolbox for plants, ACS synthetic biology 3, 839-843. Duportet, X., Wroblewska, L., Guye, P., Li, Y., Eyquem, J., Rieders, J., Rimchala, T., Batt, G., and Weiss, R. (2014) A platform for rapid prototyping of synthetic gene networks in mammalian cells, Nucleic acids research 42, 13440-13451. Weber, E., Gruetzner, R., Werner, S., Engler, C., and Marillonnet, S. (2011) Assembly of designer TAL effectors by Golden Gate cloning, PLoS One 6, e19722. ACS Paragon Plus Environment

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431

16.

17.

18.

19. 20.

21. 22. 23. 24.

25.

26.

27. 28. 29.

30. 31.

32.

33.

Page 14 of 15

Robinett, C. C., Straight, A., Li, G., Willhelm, C., Sudlow, G., Murray, A., and Belmont, A. S. (1996) In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition, J Cell Biol 135, 1685-1700. Lau, I. F., Filipe, S. R., Soballe, B., Okstad, O. A., Barre, F. X., and Sherratt, D. J. (2003) Spatial and temporal organization of replicating Escherichia coli chromosomes, Mol Microbiol 49, 731-743. Matzke, A. J., Huettel, B., van der Winden, J., and Matzke, M. (2005) Use of two-color fluorescence-tagged transgenes to study interphase chromosomes in living plants, Plant Physiol 139, 1586-1596. Michaelis, C., Ciosk, R., and Nasmyth, K. (1997) Cohesins: chromosomal proteins that prevent premature separation of sister chromatids, Cell 91, 35-45. Gordon, G. S., Sitnikov, D., Webb, C. D., Teleman, A., Straight, A., Losick, R., Murray, A. W., and Wright, A. (1997) Chromosome and low copy plasmid segregation in E. coli: visual evidence for distinct mechanisms, Cell 90, 1113-1121. Dworkin, J., and Losick, R. (2002) Does RNA polymerase help drive chromosome segregation in bacteria?, Proc Natl Acad Sci U S A 99, 14089-14094. Mettrick, K. A., and Grainge, I. (2016) Stability of blocked replication forks in vivo, Nucleic acids research 44, 657-668. Thanbichler, M., and Shapiro, L. (2006) MipZ, a spatial regulator coordinating chromosome segregation with cell division in Caulobacter, Cell 126, 147-162. Wang, X., Montero Llopis, P., and Rudner, D. Z. (2014) Bacillus subtilis chromosome organization oscillates between two distinct patterns, Proc Natl Acad Sci U S A 111, 1287712882. Casini, A., Christodoulou, G., Freemont, P. S., Baldwin, G. S., Ellis, T., and MacDonald, J. T. (2014) R2oDNA designer: computational design of biologically neutral synthetic DNA sequences, ACS synthetic biology 3, 525-528. Iverson, S. V., Haddock, T. L., Beal, J., and Densmore, D. M. (2016) CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology, ACS synthetic biology 5, 99-103. Bernard, P. (1996) Positive selection of recombinant DNA by CcdB, Biotechniques 21, 320323. Santos, C. N., Regitsky, D. D., and Yoshikuni, Y. (2013) Implementation of stable and complex biological systems through recombinase-assisted genome engineering, Nat Commun 4, 2503. Bentley, W. E., and Quiroga, O. E. (1993) Investigation of subpopulation heterogeneity and plasmid stability in recombinant Escherichia coli via a simple segregated model, Biotechnol Bioeng 42, 222-234. Datsenko, K. A., and Wanner, B. L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, Proc Natl Acad Sci U S A 97, 6640-6645. Cermak, T., Doyle, E. L., Christian, M., Wang, L., Zhang, Y., Schmidt, C., Baller, J. A., Somia, N. V., Bogdanove, A. J., and Voytas, D. F. (2011) Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting, Nucleic acids research 39, e82. Waldminghaus, T., Weigel, C., and Skarstad, K. (2012) Replication fork movement and methylation govern SeqA binding to the Escherichia coli chromosome, Nucleic acids research 40, 5465-5476. Messerschmidt, S. J., Kemter, F. S., Schindler, D., and Waldminghaus, T. (2015) Synthetic secondary chromosomes in Escherichia coli based on the replication origin of chromosome II in Vibrio cholerae, Biotechnol J 10, 302-314.


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Design and assembly of DNA sequence libraries for chromosomal insertion in bacteria based on a set of modified MoClo vectors Daniel Schindler*, Sarah Milbredt*, Theodor Sperlea* and myself (* equal contribution).

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Design and Assembly of DNA Sequence Libraries ... - ACS Publications

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